Trans blot turbo rta mini pvdf transfer kit

Proteins separated by SDS-PAGE were transferred to a PVDF membrane using the Trans-Blot Turbo RTA Mini PVDF Transfer Kit (Bio-Rad Laboratories, Inc). Membranes after activating with methanol were blocked with bovine serum albumin (Amresco, LLC). For Strep-Tag II detection, HRP-conjugated StrepMAB-Classical MAb (IBA Life Sciences) was used at 1:3000 dilution in PBS. Purified mAbs, CH58 and CH59 were used as primary antibodies at 1:5000 dilution in PBS and rabbit anti-human Ab HRP conjugate (Santa Cruz Biotechnology) was used as secondary antibodies at 1:10,000 dilution in PBS. Signal from HRP-conjugated antibodies was detected using Clarity Western ECL Blotting substrate (Bio-Rad Laboratories, Inc). Band intensities were measured using Bio-Rad Gel Doc XR +System and Image Lab software.

To characterize the specificity of individual MαF-Ig mAb, 1 µg of purified ferret Ig or 0.5 µl of ferret serum was reduced and resolved by protein gel electrophoresis as describe previously. Protein transfer to polyvinylidene difluoride (PVDF) membranes was performed using the Trans-Blot Turbo RTA Mini PVDF transfer kit (Bio-Rad, Cat #1704272) and a Trans-Blot Turbo Blotting system (Bio-Rad, Hercules, CA, USA) according to the manufacture's instructions. The membrane was blocked with PBS + Tween 20 (0.1% v/v) (PBST) containing 5% BSA (VWR, Cat #0332) at room temperature (RT) with constant agitation. The PVDF membranes were then cut into strips and probed with 15 mL of PBST containing 0.1 µg/mL of individual MαF-Ig mAb overnight at RT. The following day, PVDF membranes were washed three times with PBST before incubation for 60 min at RT with 10 mL PBST containing horseradish peroxidase conjugated goat anti-mouse IgG1 (γ1-specific). Following extensive washing with PBS, membranes were treated with 4 mL Clarity™ Western ECL Substrate (Bio-Rad, Cat #1705060) and imaged using myECL Imager. Postacquisition analysis was performed using myImageAnalysis™ Software (ThermoFisher).

Homogeneous tumor powder was lysed in RIPA buffer (Thermo Scientific, Waltham, MA, USA) supplemented with 1% protease and phosphatase inhibitors (Thermo Scientific). Protein amount was measured with a Pierce^TM BCA protein Assay Kit (Thermo Scientific). Equal amounts of proteins were loaded onto 4–15% Mini-PROTEAN TGX^TM Precast Gels (Bio-Rad). Following electrophoresis in 1× Tris/glycine/SDS running Buffer (Bio-Rad), proteins were transferred to PVDF membranes using the Trans-Blot Turbo RTA Mini PVDF Transfer Kit (Bio-Rad) according to the vendor’s instructions. Non-specific binding was blocked by soaking the membranes in 5% BSA in tTBS (1* Tris-Buffered Saline, 0.1% Tween 20, Bio-Rad) at room temperature for 1 h.
Membranes were incubated with primary anti-HSP90, anti-LDHA (Cell Signaling, #2021S, dilution 1:1000), in tTBS-BSA 5% at 4 °C overnight, followed by incubation with anti-rabbit or anti-mouse secondary antibodies (Jackson IR) in tTBS-BSA 1% at room temperature for 1 h. Detection was performed using the SuperSignal^TM West Pico Plus Kit (Thermo Scientific) and an ImageQuant LAS 500 camera (GE Healthcare). Quantification was performed on ImageJ by measuring the integral of the optical density profile of the band of the expected molecular weight. No Background correction was performed.

Cell monolayers were incubated on ice with RIPA lysis buffer (Beyotime, P0013K) containing PMSF (Beyotime, ST506-2). Proteins were separated on 15% SDS-PAGE gels and subsequently transferred to PVDF membranes (Merck, ISEQ00010) using a Trans-Blot Turbo™ RTA Mini PVDF Transfer Kit (Bio-rad, 1704272). The membranes were then sequentially incubated with primary antibodies and peroxidase affinipure goat anti-rabbit or mouse IgG (H + L). Specific protein bands were analyzed using an ECL kit (Bio-rad, 1705060).

Adherent A375 and A375R cells were lysed in RIPA buffer (Thermo Scientific) supplemented with 1% protease and phosphatase inhibitors (Thermo Scientific). Protein amount in whole cell lysates was measured with a Pierce™ BCA Protein Assay Kit (Thermo Scientific). Equal amounts of protein were loaded onto 4%‐15% Mini‐PROTEAN^® TGX™ Precast Gels (Bio‐Rad). Following electrophoresis in 1× Tris/glycine/SDS running buffer (Bio‐Rad), proteins were transferred to PVDF membranes using the Trans‐Blot^® Turbo™ RTA Mini PVDF Transfer Kit (Bio‐Rad) according to the vendor's instructions. Non‐specific binding was blocked by soaking the membranes in 5% BSA in tTBS (1× Tris‐Buffered Saline, 0.1% Tween 20, Bio‐Rad) at room temperature for 1 hour.
Membranes were incubated with primary anti‐HSP90, anti‐LDHA, anti–β‐actin, anti‐HSP90, anti–c‐MYC, (Cell Signaling), anti‐MCT1, anti‐MCT4, anti‐GLUT1 (Abcam) antibodies in tTBS‐BSA 5% at 4°C overnight, followed by incubation with anti‐rabbit or anti‐mouse secondary antibodies (Jackson IR) in tTBS‐BSA 1% at room temperature for 1 hour. Detection was performed using the SuperSignal™ West Pico Plus kit (Thermo Scientific) and an ImageQuant LAS 500 camera (GE Healthcare). Quantification was performed on ImageJ by measuring the integral of the optical density profile of the band of the expected molecular weight. No background correction was performed.